We have developed methods to manipulate for the first time the natural symmetry of nucleosomes, in order to test the extent to which this symmetry is functionally important. These questions cannot be pursued in cells with natural histones. Therefore, we have designed altered histone H3s that have obligate heterodimeric interactions, and which preclude interaction with wild-type H3 molecules. We will now use these altered H3s to measure how nucleosomal asymmetry affects gene expression and histone modification patterns, as follows: Aim 1. Identify the mechanistic basis for epistatic interactions between histone tails. In our preliminary studies, we observed distinct classes of phenotypes upon mutation of modifiable residues: in one case, a single asymmetric H3 point mutation paired with a wild-type partner exhibited all the transcriptional defects of a double point mutant. In another case, genes were only misregulated in symmetric double mutants. We will extend these studies to a large set of histone mutations to understand the mechanistic basis for the epistasis observed between pairs of histone mutants. Aim 2. Determine whether histone crosstalk functions in cis or in trans. A great number of histone modifying enzymes preferentially act on nucleosomes carrying some second modification, a phenomenon often referred to as cross-talk. We will use genetic and biochemical approaches to assess whether crosstalk occurs in cis, on the same tail, or in trans, on opposite tails: we will identify the quantitative difference in gene expression between cells with cis and trans double K->R mutations in the H3 tail, and perform mass spectrometric analysis of purified asymmetric nucleosomes to determine whether second site modifications are lost in cis, in trans, or are unaffected by monomeric histone mutations. Together, these studies will reveal previously unexplored biochemical dependency pathways that alter histone modification patterns, and distinguish gene expression regulatory events that are dependent on one versus two histone H3 N-termini. Notably, because of the extreme conservation of core histones among eukaryotes, this work will open the way to exploring related questions in metazoans. Because histone modifications are central to all aspects of gene expression from yeast to man, and play major roles in human diseases including cancer, these studies will reveal unappreciated regulatory mechanisms that govern human health and growth control.